Short lifespan is one's fate, long lifespan is one's achievement: lessons from Daphnia.

Studies of longevity rely on baseline life expectancy of reference genotypes measured in standardized conditions. Variation among labs, protocols, and genotypes makes longevity intervention studies difficult to compare. Furthermore, extending lifespan under suboptimal conditions or that of a short-lived genotype may be of a lesser theoretical and translational value than extending the maximal possible lifespan. Daphnia is becoming a model organism of choice for longevity research complementing data obtained on traditional models. In this study, we report longevity of several genotypes of a long-lived species D. magna under a variety of protocols, aiming to document the highest lifespan, factors reducing it, and parameters that change with age and correlate with longevity. Combining longevity data from 25 experiments across two labs, we report a strong intraspecific variation, moderate effects of group size and medium composition, and strong genotype-by-environment interactions with respect to food level. Specifically, short-lived genotypes show no caloric restriction (CR) effect, while long-lived ones expand their lifespan even further under CR. We find that the CR non-responsive clones show little correlation between longevity and two measures of lipid peroxidation. In contrast, the long-lived, CR-responsive clones show a positive correlation between longevity and lipid hydroperoxide abundance, and a negative correlation with MDA concentration. This indicates differences among genotypes in age-related accumulation and detoxification of LPO products and their effects on longevity. Our observations support the hypothesis that a long lifespan can be affected by CR and levels of oxidative damage, while genetically determined short lifespan remains short regardless.

Differential expression of gluconeogenesis-related transcripts in a freshwater zooplankton model organism suggests a role of the Cori cycle in hypoxia tolerance.

Gluconeogenesis (GNG) is the process of regenerating glucose and NAD+ that allows for continued ATP synthesis by glycolysis during fasting or in hypoxia. Recent data from C. elegans and crustaceans challenged with hypoxia show differential and tissue-specific expression of GNG-specific genes. Here we report differential expression of several GNG-specific genes in the head and body of a model organism, Daphnia magna, a planktonic crustacean, in normoxic and acute hypoxic conditions. We predict that GNG-specific transcripts will be enriched in the body, where most of the fat tissue is located, rather than in the head, where the tissues critical for survival in hypoxia, the central nervous system and locomotory muscles, are located. We measured the relative expression of GNG-specific transcripts in each body part by qRT-PCR and normalized them by either the expression of a reference gene or the rate-limiting glycolysis enzyme pyruvate kinase (PK). Our data show that of the three GNG-specific transcripts tested, pyruvate carboxylase (PC) showed no differential expression in either the head or body. Phosphoenolpyruvate carboxykinase (PEPCK-C), on the other hand, is upregulated in hypoxia in both body parts. Fructose-1,6-bisphosphatase (FBP) is upregulated in the body relative to the head and upregulated in hypoxia relative to normoxia, with a stronger body effect in hypoxia when normalized by PK expression. These results support our hypothesis that Daphnia can survive hypoxic conditions by implementing the Cori cycle, where body tissues supply glucose and NAD+ to the brain and muscles, enabling them to continuously generate ATP by glycolysis.

Diet effects on longevity, heat tolerance, lipid peroxidation and mitochondrial membrane potential in Daphnia.

The dietary supply of polyunsaturated fatty acids (PUFA) crucially affects animals' performance at different temperatures. However, the underlying physiological mechanisms are still insufficiently understood. Here, we analyzed lifespan and heat tolerance of four genotypes of Daphnia magna reared on either the green alga Scenedesmus obliquus that lacks long-chain (> C18) PUFA, or the heterokont alga Nannochloropsis limnetica that contains C20 PUFA, both either at saturating and near-starvation levels. A significant genotype-by-diet interaction in lifespan was observed at saturating diets. The C20 PUFA-rich diet eliminated differences in lifespan among genotypes on the PUFA-deficient diet. Corrected for body length, acute heat tolerance was higher at low than at high food concentration, at least in the older of the two age groups analyzed. Genotypes differed significantly in heat tolerance, but there were no genotype-by-diet interactions. As predicted, the C20 PUFA-rich diet resulted in higher lipid peroxidation (LPO) and a lower mitochondrial membrane potential (ΔΨm). LPO levels averaged across clones and rearing conditions were inversely related to acute heat tolerance. Yet, heat tolerance was higher on the PUFA-rich diet than on the PUFA-deficient diet, particularly in older Daphnia, indicating that the C20 PUFA-rich diet allowed Daphnia to compensate for higher LPO. In contrast, Daphnia with intermediate levels of ΔΨm showed the lowest heat tolerance. Neither LPO nor ΔΨm explained the diet effects on lifespan. We hypothesize that antioxidants present in the PUFA-rich diet may have enabled higher heat tolerance of Daphnia despite higher LPO, which may also explain the lifespan expansion of otherwise short-lived genotypes.

The interplay between prior selection, mild intermittent exposure, and acute severe exposure in phenotypic and transcriptional response to hypoxia.

Hypoxia has profound and diverse effects on aerobic organisms, disrupting oxidative phosphorylation and activating several protective pathways. Predictions have been made that exposure to mild intermittent hypoxia may be protective against more severe exposure and may extend lifespan. Here we report the lifespan effects of chronic, mild, intermittent hypoxia, and short-term survival in acute severe hypoxia in four clones of Daphnia magna originating from either permanent or intermittent habitats. We test the hypothesis that acclimation to chronic mild intermittent hypoxia can extend lifespan through activation of antioxidant and stress-tolerance pathways and increase survival in acute severe hypoxia through activation of oxygen transport and storage proteins and adjustment to carbohydrate metabolism. Unexpectedly, we show that chronic hypoxia extended the lifespan in the two clones originating from intermittent habitats but had the opposite effect in the two clones from permanent habitats, which also showed lower tolerance to acute hypoxia. Exposure to chronic hypoxia did not protect against acute hypoxia; to the contrary, Daphnia from the chronic hypoxia treatment had lower acute hypoxia tolerance than normoxic controls. Few transcripts changed their abundance in response to the chronic hypoxia treatment in any of the clones. After 12 h of acute hypoxia treatment, the transcriptional response was more pronounced, with numerous protein-coding genes with functionality in oxygen transport, mitochondrial and respiratory metabolism, and gluconeogenesis, showing upregulation. While clones from intermittent habitats showed somewhat stronger differential expression in response to acute hypoxia than those from permanent habitats, contrary to predictions, there were no significant hypoxia-by-habitat of origin or chronic-by-acute treatment interactions. GO enrichment analysis revealed a possible hypoxia tolerance role by accelerating the molting cycle and regulating neuron survival through upregulation of cuticular proteins and neurotrophins, respectively.

Inverse Lansing Effect: Maternal Age and Provisioning Affecting Daughters' Longevity and Male Offspring Production.

AbstractMaternal age effects on offspring life history are known in a variety of organisms, with offspring of older mothers typically having lower life expectancy (the Lansing effect). However, there is no consensus on the generality and mechanisms of this pattern. We tested predictions of the Lansing effect in several Daphnia magna clones and observed clone-specific magnitude and direction of the maternal age effect on offspring longevity. We also report ambidirectional, genotype-specific effects of maternal age on the propensity of daughters to produce male offspring. Focusing on two clones with contrasting life histories, we demonstrate that maternal age effects can be explained by lipid provisioning of embryos by mothers of different ages. Individuals from a single-generation maternal age reversal treatment showed intermediate life span and intermediate lipid content at birth. In the clone characterized by the "inverse Lansing effect," neonates produced by older mothers showed higher mitochondrial membrane potential in neural tissues than their counterparts born to younger mothers. We conclude that an inverse Lansing effect is possible and hypothesize that it may be caused by age-specific maternal lipid provisioning creating a calorically restricted environment during embryonic development, which in turn reduces fecundity and increases life span in offspring.

Pluripotency factors determine gene expression repertoire at zygotic genome activation.

Awakening of zygotic transcription in animal embryos relies on maternal pioneer transcription factors. The interplay of global and specific functions of these proteins remains poorly understood. Here, we analyze chromatin accessibility and time-resolved transcription in single and double mutant zebrafish embryos lacking pluripotency factors Pou5f3 and Sox19b. We show that two factors modify chromatin in a largely independent manner. We distinguish four types of direct enhancers by differential requirements for Pou5f3 or Sox19b. We demonstrate that changes in chromatin accessibility of enhancers underlie the changes in zygotic expression repertoire in the double mutants. Pou5f3 or Sox19b promote chromatin accessibility of enhancers linked to the genes involved in gastrulation and ventral fate specification. The genes regulating mesendodermal and dorsal fates are primed for activation independently of Pou5f3 and Sox19b. Strikingly, simultaneous loss of Pou5f3 and Sox19b leads to premature expression of genes, involved in regulation of organogenesis and differentiation.

Intelligent high-throughput intervention testing platform in Daphnia.

We present a novel platform for testing the effects of interventions on the life- and healthspan of a short-lived freshwater organism with complex behavior and physiology-the planktonic crustacean Daphnia magna. Within this platform, dozens of complex behavioral features of both routine motion and response to stimuli are continuously quantified over large synchronized cohorts via an automated phenotyping pipeline. We build predictive machine-learning models calibrated using chronological age and extrapolate onto phenotypic age. We further apply the model to estimate the phenotypic age under pharmacological perturbation. Our platform provides a scalable framework for drug screening and characterization in both life-long and instant assays as illustrated using a long-term dose-response profile of metformin and a short-term assay of well-studied substances such as caffeine and alcohol.

Lack of age-related respiratory changes in Daphnia.

Aging is a multifaceted process of accumulation of damage and waste in cells and tissues; age-related changes in mitochondria and in respiratory metabolism have the focus of aging research for decades. Studies of aging in nematodes, flies and mammals all revealed age-related decline in respiratory functions, with somewhat controversial causative role. Here we investigated age-related changes in respiration rates, lactate/pyruvate ratio, a commonly used proxy for NADH/NAD+ balance, and mitochondrial membrane potential in 4 genotypes of an emerging model organism for aging research, a cyclic parthenogen Daphnia magna. We show that total body weight-adjusted respiration rate decreased with age, although this decrease was small in magnitude and could be fully accounted for by the decrease in locomotion and feeding activity. Neither total respiration normalized by protein content, nor basal respiration rate measured in anaesthetized animals decreased with age. Lactate/pyruvate ratio and mitochondrial membrane potential (∆Ψmt) showed no age-related changes, with possible exceptions of ∆Ψmt in epipodites (excretory and gas exchange organs) in which ∆Ψmt decreased with age and in the optical lobe of the brain, in which ∆Ψmt showed a maximum at middle age. We conclude that actuarial senescence in Daphnia is not caused by a decline in respiratory metabolism and discuss possible mechanisms of maintaining mitochondrial healthspan throughout the lifespan.

False and true positives in arthropod thermal adaptation candidate gene lists.

Genome-wide studies are prone to false positives due to inherently low priors and statistical power. One approach to ameliorate this problem is to seek validation of reported candidate genes across independent studies: genes with repeatedly discovered effects are less likely to be false positives. Inversely, genes reported only as many times as expected by chance alone, while possibly representing novel discoveries, are also more likely to be false positives. We show that, across over 30 genome-wide studies that reported Drosophila and Daphnia genes with possible roles in thermal adaptation, the combined lists of candidate genes and orthologous groups are rapidly approaching the total number of genes and orthologous groups in the respective genomes. This is consistent with the expectation of high frequency of false positives. The majority of these spurious candidates have been identified by one or a few studies, as expected by chance alone. In contrast, a noticeable minority of genes have been identified by numerous studies with the probabilities of such discoveries occurring by chance alone being exceedingly small. For this subset of genes, different studies are in agreement with each other despite differences in the ecological settings, genomic tools and methodology, and reporting thresholds. We provide a reference set of presumed true positives among Drosophila candidate genes and orthologous groups involved in response to changes in temperature, suitable for cross-validation purposes. Despite this approach being prone to false negatives, this list of presumed true positives includes several hundred genes, consistent with the "omnigenic" concept of genetic architecture of complex traits.

To the origin of Lake Baikal endemic gammarid radiations, with description of two new Eulimnogammarus spp.

Extraordinarily diverse morphologically and ecologically, Lake Baikal's two endemic gammaroidean amphipod clades are both firmly placed within the paraphyletic genus Gammarus, based both on morphological and molecular characters. However, the exact placement of the two Baikal clades remains elusive, making reconstruction of the ancestral state of Baikal endemic radiation difficult. We sequenced 2 mitochondrial and 3 nuclear genes from several species of each of the two clades aiming to represent early branches of the radiation. We also describe two new species of Baikal gammarids, Eulimnogammarus etingovae sp. nov. and Eulimnogammarus tchernykhi sp. nov., with some morphology suggestive of basal position within the radiation. We confirm the two previously demonstrated Baikal clades, but cannot unequivocally support any of the previous hypotheses about affinities of the two Baikal clades within palearctic Gammarus species. Rather, it appears that the two Baikal endemic radiations separated from the rest of freshwater Palearctic forms early and rapidly, probably as part of gammarid diversification during colonization of fresh waters in Middle Eocene.

Excessive Parallelism in Protein Evolution of Lake Baikal Amphipod Species Flock.

Repeated emergence of similar adaptations is often explained by parallel evolution of underlying genes. However, evidence of parallel evolution at amino acid level is limited. When the analyzed species are highly divergent, this can be due to epistatic interactions underlying the dynamic nature of the amino acid preferences: The same amino acid substitution may have different phenotypic effects on different genetic backgrounds. Distantly related species also often inhabit radically different environments, which makes the emergence of parallel adaptations less likely. Here, we hypothesize that parallel molecular adaptations are more prevalent between closely related species. We analyze the rate of parallel evolution in genome-size sets of orthologous genes in three groups of species with widely ranging levels of divergence: 46 species of the relatively recent lake Baikal amphipod radiation, a species flock of very closely related cichlids, and a set of significantly more divergent vertebrates. Strikingly, in genes of amphipods, the rate of parallel substitutions at nonsynonymous sites exceeded that at synonymous sites, suggesting rampant selection driving parallel adaptation. At sites of parallel substitutions, the intraspecies polymorphism is low, suggesting that parallelism has been driven by positive selection and is therefore adaptive. By contrast, in cichlids, the rate of nonsynonymous parallel evolution was similar to that at synonymous sites, whereas in vertebrates, this rate was lower than that at synonymous sites, indicating that in these groups of species, parallel substitutions are mainly fixed by drift.

Quantitative Proteomics Reveals Remodeling of Protein Repertoire Across Life Phases of Daphnia pulex.

Although the microcrustacean Daphnia is becoming an organism of choice for proteomic studies, protein expression across its life cycle have not been fully characterized. Proteomes of adult females, juveniles, asexually produced embryos, and the ephippia-resting stages containing sexually produced diapausing freezing- and desiccation-resistant embryos are analyzed. Overall, proteins with known molecular functions are more likely to be detected than proteins with no detectable orthology. Similarly, proteins with stronger gene model support in two independent genome assemblies can be detected, than those without such support. This suggests that the proteomics pipeline can be applied to verify hypothesized proteins, even given questionable reference gene models. In particular, upregulation of vitellogenins and downregulation of actins and myosins in embryos of both types, relative to juveniles and adults, and overrepresentation of cell-cycle related proteins in the developing embryos, relative to diapausing embryos and adults, are observed. Upregulation of small heat-shock proteins and peroxidases, as well as overrepresentation of stress-response proteins in the ephippium relative to the asexually produced non-diapausing embryos, is found. The ephippium also shows upregulation of three trehalose-synthesis proteins and downregulation of a trehalose hydrolase, consistent with the role of trehalose in protection against freezing and desiccation.

Pou5f3, SoxB1, and Nanog remodel chromatin on high nucleosome affinity regions at zygotic genome activation.

The zebrafish embryo is transcriptionally mostly quiescent during the first 10 cell cycles, until the main wave of zygotic genome activation (ZGA) occurs, accompanied by fast chromatin remodeling. At ZGA, homologs of the mammalian stem cell transcription factors (TFs) Pou5f3, Nanog, and Sox19b bind to thousands of developmental enhancers to initiate transcription. So far, how these TFs influence chromatin dynamics at ZGA has remained unresolved. To address this question, we analyzed nucleosome positions in wild-type and maternal-zygotic (MZ) mutants for pou5f3 and nanog by MNase-seq. We show that Nanog, Sox19b, and Pou5f3 bind to the high nucleosome affinity regions (HNARs). HNARs are spanning over 600 bp, featuring high in vivo and predicted in vitro nucleosome occupancy and high predicted propeller twist DNA shape value. We suggest a two-step nucleosome destabilization-depletion model, in which the same intrinsic DNA properties of HNAR promote both high nucleosome occupancy and differential binding of TFs. In the first step, already before ZGA, Pou5f3 and Nanog destabilize nucleosomes at HNAR centers genome-wide. In the second step, post-ZGA, Nanog, Pou5f3, and SoxB1 maintain open chromatin state on the subset of HNARs, acting synergistically. Nanog binds to the HNAR center, whereas the Pou5f3 stabilizes the flanks. The HNAR model will provide a useful tool for genome regulatory studies in a variety of biological systems.

Temperature gradient affects differentiation of gene expression and SNP allele frequencies in the dominant Lake Baikal zooplankton species.

Local adaptation and phenotypic plasticity are main mechanisms of organisms' resilience in changing environments. Both are affected by gene flow and are expected to be weak in zooplankton populations inhabiting large continuous water bodies and strongly affected by currents. Lake Baikal, the deepest and one of the coldest lakes on Earth, experienced epilimnion temperature increase during the last 100 years, exposing Baikal's zooplankton to novel selective pressures. We obtained a partial transcriptome of Epischura baikalensis (Copepoda: Calanoida), the dominant component of Baikal's zooplankton, and estimated SNP allele frequencies and transcript abundances in samples from regions of Baikal that differ in multiyear average surface temperatures. The strongest signal in both SNP and transcript abundance differentiation is the SW-NE gradient along the 600+ km long axis of the lake, suggesting isolation by distance. SNP differentiation is stronger for nonsynonymous than synonymous SNPs and is paralleled by differential survival during a laboratory exposure to increased temperature, indicating directional selection operating on the temperature gradient. Transcript abundance, generally collinear with the SNP differentiation, shows samples from the warmest, less deep location clustering together with the southernmost samples. Differential expression is more frequent among transcripts orthologous to candidate thermal response genes previously identified in model arthropods, including genes encoding cytoskeleton proteins, heat-shock proteins, proteases, enzymes of central energy metabolism, lipid and antioxidant pathways. We conclude that the pivotal endemic zooplankton species in Lake Baikal exists under temperature-mediated selection and possesses both genetic variation and plasticity to respond to novel temperature-related environmental pressures.

Net Evolutionary Loss of Residue Polarity in Drosophilid Protein Cores Indicates Ongoing Optimization of Amino Acid Composition.

Amino acid frequencies in proteins may not be at equilibrium. We consider two possible explanations for the nonzero net residue fluxes in drosophilid proteins. First, protein interiors may have a suboptimal residue composition and be under a selective pressure favoring stability, that is, leading to the loss of polar (and the gain of large) amino acids. One would then expect stronger net fluxes on the protein interior than at the exposed sites. Alternatively, if most of the polarity loss occurs at the exposed sites and the selective constraint on amino acid composition at such sites decreases over time, net loss of polarity may be neutral and caused by disproportionally high occurrence of polar residues at exposed, least constrained sites. We estimated net evolutionary fluxes of residue polarity and volume at sites with different solvent accessibility in conserved protein families from 12 species of Drosophila. Net loss of polarity, miniscule in magnitude, but consistent across all lineages, occurred at all sites except the most exposed ones, where net flux of polarity was close to zero or, in membrane proteins, even positive. At the intermediate solvent accessibility the net fluxes of polarity and volume were similar to neutral predictions, whereas much of the polarity loss not attributable to neutral expectations occurred at the buried sites. These observations are consistent with the hypothesis that residue composition in many proteins is structurally suboptimal and continues to evolve toward lower polarity in the protein interior, in particular in proteins with intracellular localization. The magnitude of polarity and volume changes was independent from the protein's evolutionary age, indicating that the approach to equilibrium has been slow or that no such single equilibrium exists.

Antioxidant capacity, lipid peroxidation, and lipid composition changes during long-term and short-term thermal acclimation in Daphnia.

Examples of phenotypic plasticity-the ability of organisms of identical genotypes to produce different phenotypes in response to the environment-are abundant, but often lack data on the causative physiology and biochemistry. Phenotypes associated with increased protection against or reduced damage from harmful environments may, in fact, be downstream effects of hidden adaptive responses that remain elusive to experimental measurement or be obscured by homeostatic or over-compensatory effects. The freshwater zooplankton crustacean Daphnia drastically increases its heat tolerance as the result of acclimation to high temperatures, an effect often assumed to be based on plastic responses allowing better protection against oxidative stress. Using several geographically distant Daphnia magna genotypes, we demonstrate that the more heat tolerant individuals have a higher total antioxidant capacity (TAC) both in the comparison of heat-acclimated vs. non heat-acclimated females and in the comparison of females to age- and body size-matched males, which show lower heat tolerance than females. However, experimental manipulations of hypothesized antioxidant pathways by either glutathione addition or glutathione synthesis inhibition had no effect on heat tolerance. Lipid peroxidation (LPO), contrary to expectations, did not appear to be a predictive measure of susceptibility to thermal damage: LPO was higher, not lower, in more heat tolerant heat-acclimated individuals after exposure to a lethally high temperature. We hypothesize that LPO may be maintained in Daphnia at a constant level in the absence of acute exposure to elevated temperature and increase as a by-product of  a possible protective antioxidant mechanism during such exposure. This conclusion is corroborated by the observed short-term and long-term changes in phospholipid composition that included an increase in fatty acid saturation at 28 °C and up-regulation of certain long-chain polyunsaturated fatty acids. Phospholipid composition was more strongly affected by recently experienced temperature (4-day transfer) than by long-term (2 generations) temperature acclimation. This is consistent with partial loss of thermal tolerance after a short-term switch to a reciprocal temperature. As predicted under the homeoviscous adaptation hypothesis, the more heat tolerant Daphnia showed lower membrane fluidity than their less heat tolerant counterparts, in comparison both between acclimation temperatures and among different genotypes. We conclude that thermal tolerance in Daphnia is influenced by total antioxidant capacity and membrane fluidity at high temperatures, with both effects possibly reflecting changes in phospholipid composition.

Nuclear DNA content correlates with depth, body size, and diversification rate in amphipod crustaceans from ancient Lake Baikal, Russia.

Lake Baikal in Russia is a large, ancient lake that has been the site of a major radiation of amphipod crustaceans. Nearly 400 named species are known in this single lake, and it is thought that many more await description. The size and depth of Lake Baikal, in particular, may have contributed to the radiation of endemic amphipods by providing a large number of microhabitats for species to invade and subsequently experience reproductive isolation. Here we investigate the possibility that large-scale genomic changes have also accompanied diversification in these crustaceans. Specifically, we report genome size estimates for 36 species of Baikal amphipods, and examine the relationship between genome size, body size, and the maximum depths at which the amphipods are found in the lake. Genome sizes ranged nearly 8-fold in this sample of amphipod species, from 2.15 to 16.63 pg, and there were significant, positive, phylogenetically corrected relationships between genome size, body size, maximum depth, and diversification rate among these species. Our results suggest that major genomic changes, including transposable element proliferation, have accompanied speciation that was driven by selection for differences in body size and habitat preference in Lake Baikal amphipods.

Transcriptome-based phylogeny of endemic Lake Baikal amphipod species flock: fast speciation accompanied by frequent episodes of positive selection.

Endemic species flocks inhabiting ancient lakes, oceanic islands and other long-lived isolated habitats are often interpreted as adaptive radiations. Yet molecular evidence for directional selection during species flocks radiation is scarce. Using partial transcriptomes of 64 species of Lake Baikal (Siberia, Russia) endemic amphipods and two nonendemic outgroups, we report a revised phylogeny of this species flock and analyse evidence for positive selection within the endemic lineages. We confirm two independent invasions of amphipods into Baikal and demonstrate that several morphological features of Baikal amphipods, such as body armour and reduction in appendages and sensory organs, evolved in several lineages in parallel. Radiation of Baikal amphipods has been characterized by short phylogenetic branches and frequent episodes of positive selection which tended to be more frequent in the early phase of the second invasion of amphipods into Baikal when the most intensive diversification occurred. Notably, signatures of positive selection are frequent in genes encoding mitochondrial membrane proteins with electron transfer chain and ATP synthesis functionality. In particular, subunits of both the membrane and substrate-level ATP synthases show evidence of positive selection in the plankton species Macrohectopus branickii, possibly indicating adaptation to active plankton lifestyle and to survival under conditions of low temperature and high hydrostatic pressures known to affect membranes functioning. Other functional categories represented among genes likely to be under positive selection include Ca-binding muscle-related proteins, possibly indicating adaptation to Ca-deficient low mineralization Baikal waters.

Functional genomics of acclimation and adaptation in response to thermal stress in Daphnia.

BACKGROUND: Gene expression regulation is one of the fundamental mechanisms of phenotypic plasticity and is expected to respond to selection in conditions favoring phenotypic response. The observation that many organisms increase their stress tolerance after acclimation to moderate levels of stress is an example of plasticity which has been long hypothesized to be based on adaptive changes in gene expression. We report genome-wide patterns of gene expression in two heat-tolerant and two heat-sensitive parthenogenetic clones of the zooplankton crustacean Daphnia pulex exposed for three generations to either optimal (18°C) or substressful (28°C) temperature. RESULTS: A large number of genes responded to temperature and many demonstrated a significant genotype-by-environment (GxE) interaction. Among genes with a significant GxE there were approximately equally frequent instances of canalization, i.e. stronger plasticity in heat-sensitive than in heat-tolerant clones, and of enhancement of plasticity along the evolutionary vector toward heat tolerance. The strongest response observed is the across-the-board down-regulation of a variety of genes occurring in heat-tolerant, but not in heat-sensitive clones. This response is particularly obvious among genes involved in core metabolic pathways and those responsible for transcription, translation and DNA repair. CONCLUSIONS: The observed down-regulation of metabolism, consistent with previous findings in yeast and Drosophila, may reflect a general compensatory stress response. The associated down-regulation of DNA repair pathways potentially creates a trade-off between short-term benefits of survival at high temperature and long-term costs of accelerated mutation accumulation.

Faster evolving Drosophila paralogs lose expression rate and ubiquity and accumulate more non-synonymous SNPs.

BACKGROUND: Duplicated genes can indefinately persist in genomes if either both copies retain the original function due to dosage benefit (gene conservation), or one of the copies assumes a novel function (neofunctionalization), or both copies become required to perform the function previously accomplished by a single copy (subfunctionalization), or through a combination of these mechanisms. Different models of duplication retention imply different predictions about substitution rates in the coding portion of paralogs and about asymmetry of these rates. RESULTS: We analyse sequence evolution asymmetry in paralogs present in 12 Drosophila genomes using the nearest non-duplicated orthologous outgroup as a reference. Those paralogs present in D. melanogaster are analysed in conjunction with the asymmetry of expression rate and ubiquity and of segregating non-synonymous polymorphisms in the same paralogs. Paralogs accumulate substitutions, on average, faster than their nearest singleton orthologs. The distribution of paralogs' substitution rate asymmetry is overdispersed relative to that of orthologous clades, containing disproportionally more unusually symmetric and unusually asymmetric clades. We show that paralogs are more asymmetric in: a) clades orthologous to highly constrained singleton genes; b) genes with high expression level; c) genes with ubiquitous expression and d) non-tandem duplications. We further demonstrate that, in each asymmetrically evolving pair of paralogs, the faster evolving member of the pair tends to have lower average expression rate, lower expression uniformity and higher frequency of non-synonymous SNPs than its slower evolving counterpart. CONCLUSIONS: Our findings are consistent with the hypothesis that many duplications in Drosophila are retained despite stabilising selection being more relaxed in one of the paralogs than in the other, suggesting a widespread unfinished pseudogenization. This phenomenon is likely to make detection of neo- and subfunctionalization signatures difficult, as these models of duplication retention also predict asymmetries in substitution rates and expression profiles.